This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

下面所描述的协议如下UIUC人类研究伦理委员会的指导方针。 1.收集和外科组织消解<ol><li>收集后右结肠切除术（ 图1A和1B）的肿瘤组织样品。收集来自邻近部位的健康组织为好。 </li><li>马上转移的组织，以15毫升小瓶含12毫升HBSS解决方案。继续冰瓶内的绝缘泡沫箱。 </li><li>传送后45分钟的含有组织小瓶组织培养罩作进一步的处理。保持罩内的冰块上的小瓶。 </li><li>倒入供给组织成含有6-7毫升的HBSS溶液用移液管6孔板。 注意:每孔组织的量不在这个漂洗步骤的关键。 </li><li>保持一个冰块在6孔板。在冲洗液HBSS组织两次。 </li><li>干净切割组织与无菌分离SCIS节SOR。百果组织成更小的部分不超过1-2毫米3大小用无菌手术刀片更大。 </li><li>称重经标记的0.1％胰蛋​​白酶小瓶（含有1毫升0.1％的胰蛋白酶溶液）转移所述组织之前。转移〜20 mg组织到每个所述的0.1％胰蛋​​白酶的小瓶用吸管的。 </li><li>尽量减少的HBSS转移到胰蛋白酶小瓶，以避免进一步的稀释胰蛋白酶。 </li><li>权衡组织转移后的胰蛋白酶小瓶;文件的差异作为组织肿块。 </li><li>倾〜20毫克组织和1ml胰蛋白酶于24孔板的每个孔中。 </li><li>把24孔平板在摇床上于冰箱，在4℃下进行16-20小时。在此等待期间或更早的时候，准备聚丙烯酰胺凝胶基质和如在第3节描述的ECM的蛋白质功能化他们。 </li></ol>组织样本单细胞悬液2酶解和ECM功能化的弹性Substrat培养分离细胞ES和刚性聚苯乙烯餐具<ol><li>后在摇床胰酶以及在4℃下16-20小时温育的，从4℃的胰蛋白酶转移组织至4℃完全生长培养基小瓶（含有2-3ml的完全生长培养基的）用移液管。保证胰蛋白酶最小转移到生长介质。 注意:完全生长培养基配方如下:补充有马血清为10％和青霉素 - 链霉素至总溶液的1％的最终浓度的RPMI 1640基础培养基中。 </li><li>将包含媒体的小瓶中的温水浴，在37℃下进行12-15分钟的组织。 </li><li>转移组织到15ml小瓶中含有用吸管的HBSS溶液，轻轻摇动。 </li><li>重复2.3。 </li><li>从HBSS溶液，并转移到0.1％的胶原酶溶液（在HBSS中）含有1ml溶液小瓶去除组织。孵育45分钟，在37℃和5％CO 2的环境中的潮湿的细胞培养箱中。在等待期间，温growt在37℃] C H媒体在15ml小瓶在水浴中。 </li><li>所有的组织碎片拔出吸管将胶原酶最小化交叉污染。喷射刚组织碎片进入预热培养基瓶中。 </li><li>放置一个40μm的良好的插入滤波器入6孔板的每个孔中。滋润过滤用1毫升细胞培养基。 </li><li>磨碎组织在媒体用10毫升玻璃吸管数次，直至组织被完全离解。 </li><li>保持枪头尽可能接近到小瓶的底部。 </li><li>存款的解决方案到过滤，清除杂物及未解离部分。 </li><li>离心机过滤的细胞悬浮液中的媒体，在150×g离心5分钟，在RT。 </li><li>事后去除上清，重悬细胞沉淀用2毫升的新鲜培养基。 </li><li>计数使用血球（如果需要）的细胞。种子被隔绝在细胞外基质（ECM）官能化的凝胶和聚苯乙烯菜肴所需浓度的原代细胞。</li></ol> 3.准备和变刚度聚丙烯酰胺（PA）凝胶和聚苯乙烯基质功能化注意:对于视觉第3节的示范，观众/读者可以参考最近的杂志可视化实验（朱庇特）第17条。 <ol><li>采用协议公布的18，19，激活12毫米2玻璃盖玻片化学确保共价水凝胶结合。 <ol><li>首先，请客玻璃盖玻片3- Aminopropyltrymethoxysilane（ATS）在室温下7分钟。 </li><li>用DI水漂洗完全除去ATS和治疗盖玻片，用0.5％戊二醛（从70％戊二醛原液在PBS中稀释）处理30分钟溶液。 </li></ol></li><li>通过将5％w / v的丙烯酰胺溶液和0.05％的N，在10mM的HEPES缓冲盐水20 -N&#39;甲基双丙烯酰胺（BIS）溶液得到2千帕凝胶溶液。使用8％w / v的丙烯酰胺和0.13％的N，在10mM的HEPES缓冲盐水-N&#39;甲基双丙烯酰胺（BIS）溶液为10千帕的凝胶20。 <ol><li>在这两种情况下，使用1:200的过硫酸铵（10％重量/体积）和1:2000 N，N，N&#39;，N&#39;四甲基乙二胺（TEMED），为中，引发剂和催化剂用于聚合过程分别。 </li><li>此外，添加100μl的荧光珠，以2千帕凝胶溶液作为受托标记21,22。 </li></ol></li><li>存款下降了20微升的12 平方毫米激活盖玻片预聚合物PA凝胶溶液。广场上的下降，另有12个平方毫米普通玻璃盖玻片。确保降盖玻片之间的利差，由于毛细作用。倒置三明治为2千帕的凝胶，以确保大多数的珠来对细胞培养物表面附近。 </li><li>治愈PA凝胶在室温45分钟。 </li><li>剥离使用单刃剃须刀顶盖滑。 注:在剥离，剥离从一个边缘进行的三明治。凝胶仍然附着在活化载玻片。 </li><li>使用之前储存在PBS液凝胶。 </li><li>官能PA凝胶和玻璃使用ECM分子，人纤连蛋白在50微克/毫升以下公开的方法23的浓度。 <ol><li>简单地说，孵化用2毫升纯水合肼O / N基板。 </li><li>除去水合肼和去离子水彻底冲洗衬底。 </li><li>洗，用5％乙酸在基板30分钟。 </li><li>除去乙酸和去离子水彻底冲洗衬底。 </li><li>保持浸渍于去离子水中30分钟的衬底。 </li><li>孵育氧化纤维连接蛋白35分钟的基板在50微克/毫升的浓度。 </li><li>冲洗在摇床上用PBS基板在低转速10分钟。 </li><li>电镀细胞之前孵育2-3毫升培养基所有基板在37℃下30分钟。 注:板细胞在人口稀少填充ð方式（1,000-3,000个/厘米2）。每个凝胶覆盖的玻璃滑需要被包含在35毫米的培养皿。使细胞的任何显微镜（至少O / N）之前完全遵守。 </li></ol></li></ol> 4.牵引力显微镜和免疫荧光显微测定<ol><li>牵引力显微镜分析<ol><li>暖0.25％胰蛋白酶-EDTA / 10％SDS在37℃溶液在水浴10分钟前牵引实验开始。 </li><li>除去从细胞培养培养箱一种凝胶在时间和地点上的荧光倒置显微镜阶段（32X放大倍率）。 </li><li>发现在视场的单个细胞。取出培养皿的盖子。 </li><li>取细胞的相衬图像（ 例如 ， 图3）。 </li><li>切换成像模式荧光和选择适当的过滤器。在此期间不要移动显微镜阶段或样品。 </li><li>取的荧光珠displac的图像由蜂窝牵引（ 图4C）编</li><li>加入1毫升胰蛋白酶/ SDS溶液到培养皿分离从凝胶中的细胞。在此期间不要移动显微镜阶段或样品。此外，取单元的控制图像，以确保完全除去从凝胶。 </li><li>取的珠的参考（零力）图像的单元除去后。 </li><li>对于所有其他凝胶重复步骤4.1.2-4.1.8。 </li><li>请使用ImageJ从步骤4.1.6和4.1.8每一种情况下获得的两个图像图像堆栈。要生成图像堆栈，用命令的ImageJ的下列顺序打开图片后:图像→堆叠→图像堆栈。 </li><li>为了获得位移场和牵引，使用ImageJ的插件下面公布的方法21，22获得这些插件使用下面的链接代码和详细的教程。 <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/ima</a>gejplugins。 <ol><li>对齐用的"模板匹配"插件堆栈中的图像。使用以下工作步骤4.1.10生成的图像堆栈后序列中的ImageJ命令:在堆栈插件→模板匹配→对齐→切片确定。保存图像堆栈因此。在下面的步骤使用这个新的图像堆栈。 </li><li>获得使用的PIV（粒子图像测速）插件（ 图4D）的位移场。使用下面的打开保存在步骤4.1.11.1图像堆栈后命令序列:插件→PIV→迭代PIV（基本）→OK→接受这个PIV和输出→确定。在同一目录下的PIV输出保存为原始图像堆栈。以此为输入下一个步骤。 </li><li>最后使用FTTC（傅立叶变换牵引术）的插件，以获得牵引力地图（ 图4E）。命令使用以下顺序:→插件→FTTC插件ERT的材料特性， 即 ，杨氏模量和PA的凝胶→确定→泊松比选择保存在步骤4.1.11.2→OK了PIV输出文件。下次FTTC在同一目录中的结果如在图像堆栈和PIV输出。 </li></ol></li></ol></li><li>免疫荧光显微镜测定<ol><li>就拿基板被免疫染色的层流罩，取出培养基。 </li><li>冲洗用PBS基板和固定细胞用PBS中的4％多聚甲醛20分钟，室温。 </li><li>为3次（每次5分钟）洗涤，用PBS基板。因此，孵育用500μl信号增强基板30分钟并漂洗用PBS。 </li><li>孵育细胞与单克隆抗粘着斑蛋白抗体以1:250稀释在PBS中在RT下45分钟。为3次（每次5分钟）洗涤，用PBS基板。 </li><li>孵育二级抗体的Alexa Fluor 488山羊抗 - 小鼠IgG的样品以1:200稀释在PBS中在RT下30分钟。为3次（每次5分钟）洗涤，用PBS基板。 </li><li>为了显现的F-肌动蛋白的结构，孵育细胞TRITC鬼笔环肽缀合物的浓度50微克/毫升，在室温45分钟。为3次（每次5分钟）洗涤，用PBS基板。 </li><li>图像（ 图5A - 5D）使用共焦扫描激光显微镜的样品。 </li></ol></li></ol>

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PLoS ONE. 5 (9), e12905 (2010).</li><li>Ulrich, T. A., de Juan Pardo, E. M., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+rigidity+of+the+extracellular+matrix+regulates+the+structure,+motility,+and+proliferation+of+glioma+cells.">The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells.</a> Cancer Res. 69, 4167-4174 (2009).</li><li>Williams, B. J., Anand, S. V., Rajagopalan, J., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-propelled+biohybrid+swimmer+at+low+Reynolds+number.">A self-propelled biohybrid swimmer at low Reynolds number.</a> Nat. Commun. 5, (2014).</li></ol>

The authors have nothing to disclose.

蜂窝牵引强调最近成为转移状态13一个潜在的生物物理指标。然而，随着原发肿瘤细胞没有实验数据的牵引文献中存在的日期。此外，还未见报道的是不同的刚度聚丙烯酰胺凝胶直接分离培养原发性结肠癌细胞。因此，我们建立对凝胶和聚苯乙烯（ 图2）优化的原发性结肠癌细胞的培养条件。在软凝胶制剂邻近细胞培养表面的荧光微珠封装允许位移场和由人原代细胞（ ?...

近年来它已成为越来越明显的是，机械微环境中，除了生物化学因素，在调节细胞功能中起重要作用。细胞可以感知并就它们所附着到（如在2D培养）或（在3D培养如）包围3-7基板刚度作出响应。通过这样做，细胞可以调节它们的分化3，形态4，迁移/蠕动5，生物物理性质6，生长7，等工序。 癌细胞也使用粘附受体的协调，分层机械化学组合和相关的信号转导的膜蛋白，细胞骨架结构，以及分子马达1,2的二维和三维矩阵刚度作出响应。例如，乳腺上皮细胞（的MECs）上为150Pa衬底培养时形成正常腺泡实质是类似于刚度健康的乳腺组织。有趣的是，它们表现出的显影肿瘤的标志，其结构和转录，当较硬基板（&gt; 5000帕），模仿一个肿瘤基质8的刚度进行培养。此外，另一实验表明，乳房肿瘤发生是伴随着胶原交联和ECM加强9。最近的实验表明，人结肠癌（HCT-8）细胞显示样表型（MLP）转移时，他们就具有生理学相关刚度（20-47千帕）2D基板培养，但不能在非常僵硬（3.6 GPA）衬底-10- 12。这些细胞的第一种形式的肿瘤样细胞团，然后相互分离，从外围开始。作为这种上皮到圆形形态（E至R转变）发生变化时，它们增殖，减少细胞 - 细胞和细胞-ECM粘附，并成为迁移的。 HCT-8细胞培养上很辛苦聚苯乙烯基板没有表现出这些恶性特征。因此，已经假定HCT-8细胞成为转移性由于其暴露于适当的微环境。值得注意的是，这些实验进行了永生化癌细胞株或鼠衍生的原代细胞，不与原代人癌细胞。 最近的一项研究提出，扩充蜂窝牵引应力可被用作一个潜在的生物物理签名为转移细胞13。研究涉及测量牵引力为在聚丙烯酰胺凝胶不同的人类肿瘤细胞株。据发现，转移性癌细胞可以发挥显著较高牵引应力相比，非转移性的细胞在所有情况下13。然而，这些结果直接矛盾对鼠衍生的乳腺癌细胞系14中的早些时候发表的研究结果。此外，最近的一项研究强调了他们的细胞骨架重构亲永生和初级人体细胞之间的显着差异蛋白分析和细胞存活蛋白表达15。因此，重新许多生物物理测定法包括牵引原发性人类癌细胞的是重要的。这将解决的问题的原代细胞是否概括永生化癌细胞系牵引趋势。 这里所描述的协议被优化用于原代人结肠细胞（包括健康的和癌性的），并为它们对软底物（聚丙烯酰胺凝胶），以及在培养皿中培养的隔离。该协议基于消化外科组织样品成单细胞悬浮液16的结果酶解。据我们所知，这是直接在软水剂基质与牵引嵌入荧光微球培养流式细胞仪分离原发性结肠癌肿瘤和正常细胞的第一个示范。透明凝胶基质也让免疫。这个实验表明在F-肌动蛋白组织和差异粘着斑在原代人结肠癌细胞作为基材硬度的变化。该细胞培养平台开辟了探索原代人细胞的各种生物物理性质，如细胞刚度和牵引作为癌症预后参数的可能性。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>HBSS</td><td>Life technologies</td><td>14175-095</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-516F</td><td></td></tr><tr><td>Trypsin</td><td>Worthington</td><td>LS003736</td><td></td></tr><tr><td>Collagenese</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>3- Aminopropyltrymethoxysilane (ATS)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>281778</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Polysciences, Inc.</td><td>01201-5</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A4058</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-methylenebisacrylamide (bis)</td><td>Sigma-Aldrich</td><td>M1533</td><td></td></tr><tr><td>HEPES buffer solution</td><td>Sigma-Aldrich</td><td>83264</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Bio-Rad</td><td>161-0700</td><td></td></tr><tr><td>Human fibronectin</td><td>BD biosciences</td><td>354008</td><td></td></tr><tr><td>Hydrazine hydrate</td><td>Sigma-Aldrich</td><td>18412</td><td>Hazardous</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>RT15710</td><td></td></tr><tr><td>Signal enhancer</td><td>Life technologies</td><td>I36933</td><td></td></tr><tr><td>Monoclonal anti vinculin antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>V9131</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG</td><td>Life technologies</td><td>A11001</td><td></td></tr><tr><td>TRITC phalloidin conjugates&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1951</td><td></td></tr><tr><td>0.1 &amp;micro;m fluroscent beads</td><td>Life technologies</td><td>F8801</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life technologies</td><td>25200-056</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>12 mm&lt;sup&gt;2 &lt;/sup&gt;glass cover slips</td><td>Corning</td><td>2865-12</td><td></td></tr></tbody></table>

isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

癌细胞响应不同癌细胞使用粘附受体组成的协调，分层机械化学系统和相关的信号转导的膜蛋白，细胞骨架结构，以及分子马达1,2与基质的机械刚度以复杂的方式。Mechanosensitivity 体外是与永生化细胞系或鼠衍生的原代细胞，不与原代人肿瘤细胞研究主要。因此，知之甚少初级人结肠癌细胞的体外 mechanosensitivity。这里，一个优化的协议被开发，描述从健康和癌外科人类组织样品的原代人结肠细胞的分离。孤立的结肠细胞，然后软（2千帕刚度）和僵硬（10千帕刚度）聚丙烯酰胺水凝胶和刚性聚苯乙烯（〜3.6 GPA刚度）由细胞外基质官能化基底（纤连蛋白培养成功在这种情况下）。荧光微珠嵌入在细胞培养物表面附近软凝胶，和牵引试验被执行以评估使用免费开放存取软件蜂窝收缩应力。此外，在不同的刚度衬底免疫荧光显微镜提供有关主细胞形态，细胞骨架组织及纽蛋白含有粘着斑作为底刚度的函数的有用信息。

上述协议被成功地用于对来自四个不同患者的多个组织样品（n = 12）下的机构审查委员会的指导方针。 图1A示出了从该组织切片的细胞培养物获得的手术后右一代表结肠直肠肿瘤。在转移到层流罩，以便进一步处理后的HBSS溶液的典型组织截面示于图1B。消化外科组织样品成单细胞悬浮液酶解的示意图示于图2。 成功的组织分离后，分离原?...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Research

JoVE Journal

Bioengineering

15.5K 次观看.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

视频: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

科研

癌症研究

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

医学

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

生物学

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

原发性患者特异性主动脉平滑肌细胞的分离和体外半定量实时收缩测量

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

Source: Ali, M. Y et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52532"> Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J. Vis. Exp. (2015)
This video describes the technique of studying the cytoskeleton and focal adhesions in primary human colon cancer cells to understand its variations depending on substrate rigidity. This culture of cancer cells on soft and hard substrates allows various downstream biophysical measurements for cancer prognosis.

细胞骨架和黏着斑组织测定:一种基于免疫荧光的研究细胞粘附和在底物上扩散的方法

Source: Ali, M. Y et al.,  Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for ...